Drs. Faguet and Rowland Reply

Faguet, Robert A.; Rowland, Kay F.

doi:10.1176/ajp.136.6.859

Cited by 2 publications

(6 citation statements)

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Section: Pha Purification and Lodinationmentioning

confidence: 99%

“…LPHA was prepared from a single batch of PHA-P (Difco Laboratories, Detroit, MI) according to a modification of the column chromatography method of Webber et al 181, as previously described [9]. This homogeneous mitogenic glycoprotein was utilized for both metabolic and receptor studies at concentrations appropriate for the purpose intended and based upon known receptor occupancy and metabolic response profiles previously reported [9,10]. For receptor studies, LPHA was iodinated (reduc-ta~~t-free'*~I, specific activity 17 Ci/mM, ICN, Life Sciences Group, Irvine, CA) by the chloramine-T method of Hunter [ll] and adjusted to a final specific activity of 150,000 CPM/pg LPHA-protein in 0.5% albumin (bovine serum albumin, Grand Island Biological Co., Grand Island, NY).…”

Section: Pha Purification and Lodinationmentioning

confidence: 99%

“…For receptor studies, LPHA was iodinated (reduc-ta~~t-free'*~I, specific activity 17 Ci/mM, ICN, Life Sciences Group, Irvine, CA) by the chloramine-T method of Hunter [ll] and adjusted to a final specific activity of 150,000 CPM/pg LPHA-protein in 0.5% albumin (bovine serum albumin, Grand Island Biological Co., Grand Island, NY). Details of the LPHA purification and iodination procedures have been published elsewhere [9]. Identity of '251-LPHA with native LPHA was ascertained by column chromatography, ultracentrifugation analysis in various density gradients, competitive binding studies, and by their biological activity judged by DNA synthesis responses of normal human lymphocytes 18-lo].…”

Section: Pha Purification and Lodinationmentioning

confidence: 99%

“…The methodology used to evaluate the binding of LPHA to specific human lymphocyte membrane receptors has been described in detail elsewhere [4,9,10]. In brief, aliquots of lo6 lymphocytes suspended in 0.1 ml of ME medium containing 0.1 % albumin (ME medium/albumin) were incubated in duplicate plastic culture tubes presoaked with 0.5% albumin in phosphate buffered saline (NaC1-Pi, pH 7.2) with LPHA concentrations ranging from 1 X low9 to 2 X 10-5M (using 12%-LPHA as a tracer) in 0.4 ml of NaC1-Pi with 0.1% albumin (NaC1-Pi/albumin).…”

Section: Characterization Of Lpha Binding To Lymphocyte Receptorsmentioning

confidence: 99%

“…Receptor-ligand complex dissociation was monitored from the radioactivity remaining on cells collected on 0.5 % albuminpresoaked 0.45 pm millipore filters after discarding the supernatants from two centrifugations at 400g for 5 min at 4°C. In some experiments, this undisturbed, basal dissociation reaction was compared to receptor-ligand dissociation in the presence of an excess of ligand, according to De Meyts et a1 [l], as previously described for this system [4,9,12]. The nonspecific binding component assessed from identical concomitant incubations containing a 100-fold excess of LPHA and from blanks containing no cells were subtracted from the total to determine receptor-specific ligand binding.…”

Section: Characterization Of Lpha Binding To Lymphocyte Receptorsmentioning

confidence: 99%

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Ligand–receptor interactions: Detailed evaluation of occupancy‐dependent affinity

Faguet

1986

J of Cellular Biochemistry

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The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses.

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Section: Pha Purification and Lodinationmentioning

confidence: 99%